Mutations in the gene encoding the stimulatory G protein of adenylyl cyclase have been identified in subjects with Albright hereditary osteodystrophy (AHO), an inherited disorder with characteristic developmental defects and in some patients resistance to hormones that stimulate cAMP production (pseudohypoparathyroidism, PHP type la). Although the molecular basis of AHO has been established, we do not fully understand the pathogenesis of the skeletal deformities and cognitive defects that characterize this disorder. Furthermore, not all subjects with deficiency of Gas have hormone resistance (pseudo- pseudohypoparathyroidism), and resistance is not seen to all hormones that utilize cAMP as a second messenger. While all subjects with PHP type la have resistance to PTH, only about half have resistance to TSH, a lesser percentage have resistance to gonadotropins, very few have been described with resistance to GHRH or ACTH, and response to ADH is reportedly normal. In order to better understand the consequences of Gas deficiency, we have established a transgenic mouse model of AHO by the targeted disruption of the murine gene encoding GAS, Gnas. A 6kb BamHI fragment of the Gnas gene, extending from 2 kb upstream of the initiator ATG to the middle of exon 2, was isolated from a 129SVJ genomic library. A targeting vector was constructed by replacing a 500 bp fragment extending from the NcoI site at the initiator ATG to an NruI site in intron 1 with the neomycin resistance gene (neoR) . ES ceus (J1) were transfected by electroporation, and selected in 250 mg/ml G418. Southern blot analysis with oligonucleotide probes upstream of the 5' BamHI identified an additional band of the expected in 7 colonies. Three of these were shown to have an approximately 50% reduction in the levels of Gas, mRNA and Gas protein. In response to 10-5 M Forskolin and to various doses of Isoproterenol cAMP accumulation was reduced in the ES cell lines with a targeted disruption of Gnas, relative to 5 ES cell lines with random integration of targeting vector or to normal ES cells. In 2 ES cell lines, Southern blot analysis with a probe downstream of the 3' BamHI site confirmed proper targeting of the Gnas allele, and karyotype analysis revealed a normal number of chromosomes. These were injected into C57B1/6 mouse embryos and implanted into pseudopregnant mice. One ES cell line gave only female chimera, which did not produce any progeny derived from the ES cells. Four male chimeras, from the other ES cell line, were bred with normal CD-1 females and sired 125 F, progeny, of which 28 carried the targeted Gnas allele. The only obvious phenotype was that F1 progeny with the targeted allele were smaller than their normal littermates. Matings of F1 progeny with the targeted allele with their normal siblings resulted in 65 F2 progeny of which 31 carried the targeted allele, suggesting that disruption of exon 1 of Gnas does not affect viability. F2 progeny will be used to study expression of Gas in various tissues, resistance to hormones that stimulate the production of cAMP, and skeletal development.